Heme-Based Oxygen Sensor Histidine Kinase Afgchk and Its Intra- and Interprotein Signal Transduction Observed By Hydrogen/Deuterium Exchange and Crystallography

Abstract

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 (the intraprotein or interdomain signal transduction). Subsequently, this phosphate is then transferred to Asp52 or Asp169 of the RR protein (interprotein or protein-protein signal transduction). Several biochemical approaches were utilized in order to study both the intra- and interprotein signal transduction of AfGcHK and its two-component system: (i) enzyme kinetic study, (ii) hydrogen/deuterium exchange experiments associated with mass spectrometry, (iii) cross-linking studies, (iv) analytical ultracentrifugation and (v) X-ray crystallography. Regarding the interprotein signal transduction we have found that AfGcHK and the RR protein form a complex with 2:1 stoichiometry utilizing the analytical ultracentrifugation. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) studies on the AfGcHK:RR complex showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein’s Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Our results on intraprotein signal transduction indicate that the coordination and oxidation state of the sensor domain’s heme iron center profoundly affect the functional domain’s catalytic activity because they modulate its ATP affinity and thus change its k cat/K m ATPvalue. The contact area between the sensor and functional domains was identified by hydrogen/deuterium exchange experiments in conjunction with mass spectrometry and cross-linking studies. Moreover, X-ray crystallography revealed that the structure of the sensor domain when the heme iron complex is in the ferric state (which is associated with an active functional domain) differs from that when the complex is in the ferrous state (which is associated with an inactive functional domain). All the above mentioned findings will be discussed to illustrate the mechanism of both intra- and interprotein signal transduction in this globin-coupled histidine kinase as a representative of heme-containing oxygen sensor proteins in general.